Soybean lipoxygenase-1 oxygenates synthetic polyenoic fatty acids with an altered positional specificity: evidence for inverse substrate alignment.

Abstract

The positional specificity is the decisive enzyme property for classification of lipoxygenases and for the currently used lipoxygenase nomenclature. It has been reported before that soybean lipoxygenase-1, which oxygenates polyenoic fatty acids at alkaline pH to the corresponding n - 6 hydroperoxy derivative, exhibits a different positional specificity when either the reaction conditions or the substrate structure is altered. To investigate the impact of structural substrate modifications on the positional specificity of this enzyme and to force an inverse substrate binding, we synthesized arachidonic acid analogues modified at the omega-terminus. Care was taken that the double bond system remained unchanged so that hydrogen abstraction from all three bisallylic methylenes was theoretically possible. We found that omega-modification of arachidonic acid leads to an impaired substrate affinity and a reduced reaction rate, but we did not detect any 5-lipoxygenation products, suggesting that structural modification of the omega-end may not be sufficient to force an inverse substrate orientation. However, when both ends of the fatty acid chain (omega-terminus and free carboxylate) were modified simultaneously, a considerable share of 5-lipoxygenation products was detected. These results indicate that introduction of polar or bulky groups at the methyl terminus of polyenoic fatty acids was not sufficient to force an inverse substrate orientation. However, simultaneous introduction of an omega-OH group and methylation of the carboxylate led to formation of significant 5-lipoxygenation products, suggesting an inverse head to tail substrate orientation.